Damage induced senescent immune cells regulate regeneration of the zebrafish retina

Abstract

Purpose : Zebrafish have the innate ability to regenerate lost or damaged retinal neurons, a process that mammals are unable to recapitulate even though they possess the same cellular populations as fish. Thus, identifying potential variable factors influencing regeneration is necessary to understand the inter-species variability in regenerative potential. Senescence and its associated secretory phenotype (SASP) have been shown to influence tissue microenvironment and regeneration in fish spinal cord injury models, but the role of senescence in retina regeneration is not well understood after damage

Methods : We injected wild type AB zebrafish with NMDA to selectively damage retinal ganglion cells (RGCs) and then treated those fish with the senolytics Metformin, ABT-263, or both. We then immunostained for senescence associated beta-galactosidase (SA-BGal) to identify senescent cells, 4c4 antibodies to identify microglia/macrophages, HuC/D antibodies to identify RGCs, and injected EdU to identify proliferating cells. This allowed us to detect senescent cells and the effect of senolytics on proliferation and regeneration

Results : Intravitreal injection of NMDA resulted in transient detection of senescent cells starting at 2dpi and peaking around 12dpi, with subsequent clearance observed towards 18dpi. These senescent cells co-localized with immune cell markers. Senolytic treatment decreased senescent cell numbers at both 5 and 10dpi, and their addition decreased the number of EdU⁺ proliferating cells in the retina after damage. Analysis of the RGC layer showed that senolytic treatment resulted in visible gaps in the RGC layer, indicating less regeneration of the RGCs after damage

Conclusions : Senescent cells show a temporally regulated pattern after NMDA damage and appear to be either macrophages or microglial in nature. Transient appearance and clearance of senescent cells aligns with previous work in spinal cord injury models in zebrafish. The decrease in proliferation and increase in the number of RGC breaks after senolytic treatment supports the hypothesis that modulation of immune responses are needed for precise proper retina regeneration in zebrafish. Future experiments are needed to determine the precise mechanism for senescent cell contribution to regeneration, and how senescence impacts Muller Glia during regeneration. Our data are the first to show a pro-regenerative role for senescent cells in the retina

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.